I. PLASMINOGEN AND PLASMINOGEN ACTIVATORS
The serum protein, plasminogen, plays an integral role in the proteolytic dissolution (or fibrinolysis) of blood clots. Plasminogen is an inactive "proenzyme." It has a specific affinity for fibrin, and thus becomes incorporated into blood clots as they form. Plasminogen's proteolytic activity is released by "plasminogen activators" ("PA") that specifically cleave the molecule to yield the active protease, plasmin. Plasmin is capable of digesting the fibrin threads of blood clots, as well as other substances involved in creating blood clots, such as fibrinogen, factor V, factor VIII, prothrombin, and factor XII (for review, see Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985), herein incorporated by reference)).
Plasmin is a serine protease, and exhibits substantial amino acid and mechanistic homology with trypsin, chymotrypsin, and pancreatic elastase. Plasmin has a relatively broad trypsin-like specificity, hydrolyzing proteins and peptides at lysyl and arginyl bonds (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)).
Two classes of natural mammalian plasminogen activators have been described: urokinase-type plasminogen activator and tissue-type plasminogen activator ("t-PA") (Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985); Devlin, et al., PCT appl. WO88/05081; Kasaia et al., U.S. Pat. No. 5,098,840; Hayashi, S. et al., U.S. Pat. No. 4,851,345; Sasaki et al., U.S. Pat. No. 4,258,030; Hayashi, S. et al., U.S. Pat. No. 5,004,609; Pyke, C. et al., Amer. J. Pathol. 138:1059-1067 (1991); Madison, E. L. et al., Nature 339:721-724 (1989); Blasi, F. et al., J. Cell. Biol. 104:801-804 (1987)). These two classes of molecules can be distinguished immunologically, by tissue localization, and by the stimulation of their activity by fibrin. In addition, a third plasminogen activator, streptokinase, has also been described. Streptokinase differs from urokinase and tPA in that it is a bacterial protein produced by the streptococci.
Urokinase-type plasminogen activator (UK) is a multi-domain protein with one domain being a trypsin-like serine protease (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985); Stra.beta.burger, W. et al., FEBS Lett. 157:219-223 (1983)). This protease domain converts plasminogen to plasmin by cleavage at an arginyl residue (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)). The amino acid sequence and three-dimensional structure of several serine proteases, including trypsin, chymotrypsin, and elastase have been deduced (Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985); Stra.beta.burger, W. et al., FEBS Lett. 157:219-223 (1983)).
Urokinase is synthesized in the kidneys, and can be recovered from urine. It is initially produced as a single chain protein, "prourokinase" that can be proteolytically cleaved by plasmin into an active two-chain protein (Devlin, et al., PCT appl. WO88/05081).
Tissue-type plasminogen activator (t-PA) is produced by the cells that line the lumen of blood vessels or endothelial cells. Like urokinase, tPA is also initially produced as a single-chain molecule (Rijken, D. G. et al., J. Biol. Chem. 256:7035-7041 (1981); Pennica, D. et al., Nature 301:214-221 (1983)).
The known plasminogen activators differ significantly in characteristics such as their biological half-lives and their preference for fibrin. All three classes of activators have been widely used as thrombolytic agents for the treatment of thrombosis in myocardial infarction, stroke, arterial occlusion, etc. (Kasai et al., U.S. Pat. No. 5,098,840; Hayashi et al., U.S. Pat. No. 5,004,609; Hayashi et al., U.S. Pat. No. 4,851,345; Sasaki et al., U.S. Pat. No. 4,258,030).
The administration of t-PA for the treatment of thrombosis in myocardial infarction, stroke, arterial occlusion, and other cardiovascular diseases reflects the production of minute blood clots which are formed during the disease process. The presence of such clots significantly increases the criticality of the disease, and increases its morbidity. Since t-PA is able to activate plasminogen to plasmin, it is capable of initiating the cascade of events needed to dissolve undesired blood clots. As such, its administration significantly decreases the mortality associated with myocardial infarction and other acute cardiovascular conditions.
Unfortunately, the use of t-PA and streptokinase has been associated with the occurrence of hemorrhages in some individuals (Pendlebury, W. W. et al., Annls. Neurol. 28:210-213 (1989); Wijdicks, E. F. M. et al., Stroke 24:554-557 (1993); Kase, C. S. et al., Annls. Intern. Med. 112:17-21 (1990); Molinari, G. F. Stroke 24:523-526 (1993);), particularly when administered with anti-clotting factors such as coumarin or heparin. This phenomenon has limited the use of t-PA and streptokinase to treat cardiovascular disease in certain classes of patients, notably, the elderly (Topol, E. J. et al., New Engl. J. Med. 327:45-47 (1992); De Jaegere, P. P. et al., J. Amer. Col. Cardiol. 19:289-294 (1992); Gore, J. M. et al., Circulation 183:448-459 (1991)).